A lytic enzyme cocktail from Streptomyces sp. B578 for the control of lactic and acetic acid bacteria in wine.

Beside yeasts, lactic acid bacteria (LAB) are the most abundant microbes in must during vinification. Whereas Oenococcos oeni is commercially used as a starter culture for the biological acid reduction in wines, other species are responsible for different types of wine spoilage. Members of the genera Pediococcus, Weissella, Leuconostoc, and Lactobacillus are producers of exopolysaccharide slimes, biogenic amines, acetic acid, and other off-flavors. In order to control microbial growth, different procedures such as heating of must and addition of sulfite or lysozyme from egg white are generally applied. Yet, because of health risks, the application of sulfite should be reduced and lysozyme is not effective against all LAB. In this study, we describe exoenzymes from a Streptomyces sp. strain B578 lysing nearly all wine-relevant strains of LAB and Gram-negative acetic acid bacteria. The lytic enzymes were active under wine-making conditions, such as the presence of sulfite and ethanol, low temperatures, and low pH values. The analysis of the exoenzyme composition revealed a synergistic action of different cell wall hydrolases. In conclusion, the lytic cocktail of Streptomyces sp. B578 is a promising tool for the control of wine-spoiling bacteria.

Environmental conditions impinge on dragline silk protein composition.

The silk formed in the major ampullate (MA) gland of the orb weaving spider Nephila clavipes is composed of two silk fibroins, which are called major ampullate spidroins 1 (MaSp1) and 2 (MaSp2). Analysis of proteolytic peptides and reactivity to spidroin type specific antibodies indicated that MaSp2 constituted only a minor part in the spinning dope as well as in the spun filaments. Upon starvation, a change in the silk's characteristic features was observed that was concomitant of a decrease in the contribution of MaSp2. The silk became less elastic and stiffer, which will better tailor its usability for the safety line, albeit at the expense of its employment as the web frame threads. In addition, since MaSp2 production requires greater ATP consumption, such a shift in the protein ratio cuts down on the energy costs to produce the silk. From this change in protein composition the spider might therefore benefit twice, by synthesizing 'cheaper' silk that into the bargain has properties that potentially can better support foraging in times of food shortage.

Lymphocyte reactivity in normal and malignant cell proliferation against a phylogenetically conserved antigen in fetal extracts.

Lymphocyte reactivity to 3-M KCl extracts from fetuses of different species of origin was shown by the macrophage electrophoretic mobility (MEMB) and/or the leukocyte migration inhibition tests in tumor-bearing mice and humans. In chemical carcinogenesis, reactivity was detectable before tumors developed. Six weeks after sc injection of 1,000 micrograms benzo[a]pyrene [(BP) CAS: 50-32-8] into XVII/Bln mice, the MEMB test became positive. The latent period was 15 weeks after 1.0 micrograms BP, indicating a dose-response relationship of the phenomenon. Painting of mouse skin with 7,12-dimethylbenz[a]anthracene [(DMBA) CAS: 57-97-6], croton oil (CAS: 8001-28-3), or benzene (CAS: 71-43-2) had the same sensitizing effect as BP. In contrast to the strong carcinogens BP and DMBA that caused lymphocyte reactivity to persist until tumors developed, benzene and the promoter croton oil induced only a transient effect. Termination of treatment abolished reactivity within 12 weeks. Lymphocyte reactivity to fetal extract in normal cell proliferation was evident from the fact that two-thirds-hepatectomized rats and BCG-treated mice became MEMB-positive. In hepatectomized rats the effect was reversible according to the completion of liver regeneration. Lymphocytes from tumor-bearing mice reacted with mouse and human fetal extract as well as with extracts from different developmental stages of frogs and fish. Fetal extracts were assumed to contain a phylogenetically conserved antigen.

Reactivity in neoplasia, preneoplasia, and pregnancy of lymphocytes against fetal extracts: cross-reactions between man and mouse.

KCl extracts (3 M) from human fetuses were tested by macrophage electrophoretic mobility (MEMB), leukocyte migration inhibition, and/or leukocyte adherence inhibition techniques against lymphocytes-leukocytes from tumor-bearing humans and control persons. A positive MEMB test was found in 209 of 284 (73%) patients with various types of clinically manifest tumors. Only 15 of 134 (11%) controls gave a positive reaction by the MEMB test. Leukocyte migration was significantly inhibited in 48 of 64 (75%) tumor patients, and leukocyte adherence was significantly inhibited in 40 of 50 (80%) tumor patients versus 7 of 50 (14%) and 10 of 54 (19%) in the corresponding controls. Lymphocyte reactivity to fetal extracts is also detectable in inbred XVII/Bln, CBA/Bln, and C3H/Bln mice bearing spontaneous or transplanted tumors of different etiology. Human and mouse fetal extracts gave cross-reactions. Lymphocytes from tumor-bearing mice were reactive against fetal extracts from the cow, pig, sheep, guinea pig, cat, and chicken. Mice bearing benign skin warts induced by 7,12-dimethylbenz[a]anthracene (DMBA) as well as DMBA-treated mice without visible skin lesions gave a positive MEMB test. Likewise, pregnancy is associated with lymphocyte reactivity to fetal extracts.

A correlation between thermal stability and structural features of staphylokinase and selected mutants: a Fourier-transform infrared study.

Variants of recombinant staphylokinase (Sak) were investigated by Fourier-transform infrared spectroscopy: Sak (wild type), Sak-M26A, Sak-M26L, and Sak-G34S/R36G/R43H (Sak-B). Estimation of the secondary structure and hydrogen-deuterium exchange experiments revealed the existence of fast-exchanging and strongly solvent-exposed fractions of the helical structures in the two samples Sak and Sak-M26L. These two samples are also thermally less stable with unfolding transition temperatures of 43.7 degrees C (Sak) and 43.5 degrees C (Sak-M26L), respectively. On contrast, Sak-M26A and Sak-G34S/R36G/R43H have a slower hydrogen-deuterium exchange, have a smaller solvent-exposed portion of the helical part, and are more resistant against thermal unfolding; the transition temperatures are 51.7 degrees C and 59.3 degrees C, respectively. The secondary structure analysis was performed by two different approaches, by curve-fitting after band narrowing and by pattern recognition (factor analysis) based upon reference spectra of proteins with known crystal structure. Within the limits of the used methods, we are unable to detect significant differences in the secondary structure of the four variants of Sak. According to the results of the factor analysis, the portions of secondary structure elements were obtained to 16-20% alpha-helix, 28-30% beta-sheet, 23-27% turns, 28-30% irregular (random) and other structure. The sharp differences in the specific plasminogen-activating capacity (Sak, Sak-G34S/R36G/R43H and Sak-M26L are fully active, but Sak-M26A does not form a stable complex with plasminogen) are not reflected in the structural features revealed by the infrared spectra of this study.

Functional properties of recombinant staphylokinase variants obtained by site-specific mutagenesis of methionine-26.

Variants of recombinant staphylokinase (Sak) were produced by site-specific mutagenesis of the unique Met-26 residue and purified to homogeneity from the cell extract of transformed E. coli. The desired mutations were confirmed by cDNA and amino-acid sequence analysis. Sak-M26L, Sak-M26C, Sak-M26R, Sak-M26V and Sak-M26A were selected for further analysis on the basis of their plasminogen activating activity. The specific fibrinolytic activities of Sak-M26L, Sak-M26C and Sak were comparable (76,000 +/- 10,000, 75,000 +/- 2400 and 78,000 +/- 9700 HU/mg, respectively; mean +/- S.E., n = 3 or 4). Active site exposure in equimolar (4.5 microM) mixtures plasminogen at room temperature was more rapid with Sak-M26L than with Sak (quantitative exposure within 4 min and 8 min, respectively). Activation of 1 microM plasminogen by catalytic amounts (5 nM) of Sak-M26L initially appeared to be somewhat faster, but comparable 50 to 60% activation was obtained within 30 min. In contrast, Sak-M26R and Sak-M26V were virtually inactive, did not form active complexes with plasminogen and did not activate plasminogen. The catalytic efficiencies for plasminogen activation were comparable for plasmin-Sak-M26L, plasmin-Sak-M26C and plasmin-Sak (0.14 microM-1 s-1, 0.16 microM-1 s-1 or 0.12 microM-1 s-1, respectively). Comparable dose-dependent lysis of 0.06 ml 125I-fibrin labeled human plasma clots submerged in 0.3 ml human plasma was obtained with Sak-M26L, Sak-M26C and Sak (concentration required for 50% lysis in 2 h, EC50, of 17 +/- 1.6 nM, 19 +/- 1.4 nM and 14 +/- 2.5 nM, respectively), whereas Sak-M26R or Sak-M26V were inactive. Sak-M26A did not form a stable complex with plasminogen, as shown by gel filtration. These data establish that substitution of the unique Met residue in position 26 of the Sak sequence with Leu or Cys has little or no influence on its plasminogen activating or fibrinolytic potential. In contrast, substitution of Met-26 with either Arg or Val results in total loss of the functional activity. Thus, the amino acid in position 26 of Sak appears to be of crucial importance for the activation of plasminogen by staphylokinase.

Physical and conformational properties of staphylokinase in solution.

The structure of staphylokinase has been analyzed by solution X-ray scattering, dynamic light scattering, ultracentrifugation and ultraviolet circular dichroism spectroscopy. Staphylokinase has a radius of gyration of 2.3 nm, a Stokes radius of 2.12 nm and a maximum dimension of 10 nm. The sedimentation coefficient is 1.71 S. These physical parameters indicate that the shape of staphylokinase is very elongated. The protein molecule consists of two folded domains of similar size. The mean distance of the centres of gravity of the domains is 3.7 nm. The mutual positions of the two domains are variable in solution. Thus, the molecule is shaped like a flexible dumbbell. About 18% of the amino acids of staphylokinase are organized in helical structures, 30% are incorporated in beta-sheets and 20% form turns.

Functional significance of NH2- and COOH-terminal regions of staphylokinase in plasminogen activation.

Structure/function relationships in the activation of plasminogen with staphylokinase were studied using mutants of recombinant staphylokinase (Sak42D). Deletion of up to 10 NH2-terminal amino acids (Sak42D delta N10) did not affect plasminogen activation, but removal of 11 amino acids completely abolished the ability to activate plasminogen. Elimination of potential plasmin cleavage sites in the NH2-terminal region yielding mutants Sak42D(K8H,K10H,K11H) and Sak42D(K6H,K8H,K11H) did not alter the rate of the exposure of a proteolytically active site (amidolytic activity) in equimolar mixtures with plasminogen, but destroyed the plasminogen activator properties of these muteins. Deleting two residues following the preferred processing site at position 10 (Sak42 delta (K11,G12)) resulted in a mutein also inactive in plasminogen activation. Removal of the COOH-terminal Lys136, yielding Sak42D delta C1, or of Lys135 and Lys136 in Sak42D delta C2 resulted in proteins with strongly reduced plasminogen activation capacity. In contrast, substitution of Lys135 and Lys136 with Ala in Sak42D(K135A,K136A) did not affect activation. Cyanogen bromide cleavage of Sak42D(M26L,E61M,D82E) produced a 61 amino acid NH2-terminal and a 65 amino acid COOH-terminal fragment which did not activate plasminogen, but bound to plasminogen with affinity constants Ka of 4.0 x 10(5) M-1 and 1.4 x 10(7) M-1, respectively (as compared to a Ka of 1.1 x 10(8) M-1 for Sak42D). These results indicate that Lys11 and the COOH-terminal region of staphylokinase play a key role in the activation of plasminogen.

Synergistic cytotoxicity of human recombinant tumour necrosis factor alpha combined with microtubule effectors.

Combinations of human recombinant tumour necrosis factor alpha (rhTNF alpha) with each of four different agents disturbing the microtubule system of the cellular cytoskeleton were tested for synergistic cytotoxic action against murine melanoma B16K and L-M(S) cells. In addition to the known microtubule effectors colchicine, vincristine, and taxol, the influence of the fluorenone-azo-methine derivative alpha-diphenylene-N-(p-[bis-(beta-hydroxyethyl-amino]-phenyl)- nitrone (DHPN) on the rhTNF alpha cytotoxicity was studied. Applying a novel computer-based isobole method [Suehnel J (1990) Antiviral Res 13:23-40] concentration ranges of synergistic, zero, and antagonistic interaction were found after in vitro combination of rhTNF alpha with each of the drugs tested in a 72-h cytotoxicity assay. In contrast, a 24-h exposure of B16K cells to these combinations still did not inhibit in vitro colony formation to a greater extent than either drug alone. A preliminary in vivo experiment revealed an increased antitumour effect after treatment of established subcutaneous melanoma B16 tumours with a combination of rhTNF alpha and DHPN.

High yield production and purification of recombinant staphylokinase for thrombolytic therapy.

Recombinant plasmids were constructed in which the signal sequence of the sak42D and the sakSTAR staphylokinase genes were replaced by an ATG start codon and which express staphylokinase under the control of a tac promoter and two Shine-Dalgarno sequences in tandem. Induction of transfected E. coli TGl cells in a bacterial fermentor produced intracellular staphylokinase representing 10 to 15% of total cell protein. Gram quantities of highly purified recombinant staphylokinase were obtained from cytosol fractions by chromatography, at room temperature, on SP-Sepharose and on phenyl-Sepharose columns, with yields of 50 to 70 percent. The material, at a dose of 4 mg/kg, did not produce acute reactions or affect body weight in mice. Intravenous administration of 10 mg SakSTAR over 30 minutes in five patients with acute myocardial infarction induced complete coronary artery recanalization, without associated fibrinogen degradation. However, neutralizing antibodies appeared in the plasma of all patients within 12 to 20 days. Thus, the present expression and purification method for recombinant staphylokinase yields large amounts of highly purified mature protein (approximately 200 mg per liter fermentation broth) suitable for a more detailed clinical investigation of its potential as a thrombolytic agent.

Bizarre parosteal osteochondromatous proliferation (Nora's Lesion) of the mandible. a rare bony lesion.

Bizarre parosteal osteochondromatous proliferation (BPOP) also eponymically called "Nora's lesion", is a rare benign reactive bone lesion first reported in 1983. BPOP occurs classically on the bones of the hands and feet and long bones. This lesion can easily be confused, both clinically and microscopically, with other benign and malignant lesions of bone, including osteochondroma, parosteal osteosarcoma, myositis ossificans and reactive periostitis. BPOP has been reported to have a high rate of recurrence. Only 3 cases of BPOP of the head and neck have been reported in the literature, of which one involved the maxilla. We present a rare case of BPOP involving the mandible in a 10 year old African American male. Microscopically, a fibro-cartilaginous cap giving rise to a proliferation of variably mineralized osteophytic finger-like projections of bone was seen. Multiple trabeculae of "blue bone" were noted as well as numerous atypical appearing chondrocytes. The lesion recurred within 4 months following the initial excision but has not recurred to date after the second local excision. To the best of our knowledge, this is the first report of BPOP arising in the mandible. In addition, we discuss the clinical and microscopic features, differential diagnosis, and prognosis of this rare entity. We present a case of BPOP of the mandible and believe this is the first report of such a case in the mandible.

The prolyl cis/trans isomerase cyclophilin 18 interacts with the tumor suppressor p53 and modifies its functions in cell cycle regulation and apoptosis.

The functional diversity of the tumor suppressor protein p53 is mainly regulated by protein interactions. In this study, we describe a new interaction with the peptidyl-prolyl cis/trans isomerase cyclophilin 18 (Cyp18). The interaction reduced the sequence-specific DNA binding of p53 in vitro, whereas the inhibition of the interaction increased p53-reporter gene activity in vivo. The active site of the folding helper enzyme Cyp18 was directly involved in binding. The proline-rich region (amino acids 64-91) of p53 was most likely responsible for the observed binding because a synthetic peptide comprising amino acids 68-81 of p53 inhibited this interaction, and a p53 variant containing a proline residue at position 72 (p53(P72)) interacted with Cyp18 more effectively than the corresponding p53(R72) variant. Impairment of the Cyp18-p53 interaction induced an accumulation of cells in the G2/M phase of the cell cycle, which was more pronounced when p53(P72) was expressed compared with p53(R72) in an otherwise isogenic cellular background. Moreover, p53-dependent apoptosis was elevated in Cyp18 knockout cells, suggesting an antiapoptotic potential of Cyp18-p53 complexes. Functional in vivo data hint to a possible clinical relevance of the p53-Cyp18 interaction observed.

Single step isolation of immunofluorescence-stained, viable lymphocytes by fluorescence-activated cell sorter using ethidium bromide.

The paper presents a method for preparation of viable, immunofluorescence-stained lymphocytes in a single isolation step by fluorescence-activated cell sorter. Discrimination between viable, immunofluorescence positive cells and nonspecifically stained dead cells was carried out on the basis of the DNA-specific dye ethidium bromide. Ethidium bromide enters cells with damaged plasma membrane only. The method is described by the example of isolation of Lyt-2 positive mouse lymph node cells.

[The macrophage electrophoresis mobility (MEM) test for detection of cell sensitivity: technical analyses. I. Guinea pig peritoneal cells as indicator cells in the MEM test]. / Der Makrophagen-Elektrophorese-Mobilitäts-(MEM-) Test zum Nachweis zellulärer Sensibilisierungen: Methodische Untersuchungen. I. Peritonealzellen des Meerschweinchens als Indikatorzellen im MEM-Test.

The influence of cell selection and the time of harvesting the indicator cells (peritoneal cells of the guinea pig) on the test result was studied. In order to obtain positive test results, it is not necessary to select the cells. The time of preparation of peritoneal cells following paraffin oil application has no effect on the test result. Peritoneal cells from animals not stimulated with oil also yielded positive MEM test results. 'False negative' results in MEM studies are, to some extent, attributable to the macrophage indicator cell system.

Modulation of the spc operon affects growth and protein secretion in Bacillus subtilis.

A proximal segment of B. subtilis secY gene was placed under the control of the inducible spac promoter/Lac repressor system. This fusion was integrated into the chromosomal spc operon of B. subtilis via Campbell-like reciprocal recombination. The growth of the resulting strain was strongly IPTG dependent. With staphylokinase and alpha-amylase as reporter proteins it was found, that the protein secretion capacity of this strain was correlated to the conditions of repression or induction of the chromosomal spac promoter.

[Cross-reactivity of 3M KCl-extracts of fetal tissue from various species in the macrophage electrophoresis mobility test. II. Tissue extracts from lower vertebrates in various phases of development]. / Kreuzreaktivität von 3M KCl-Extrakten aus Fötalgewebe verschiedener Spezies im Makrophagen-Elektrophorese-Mobilitäts- (MEM-) Test. II. Extrakte aus Geweben niederer Wirbeltiere verschiedener Entwicklungsphasen.

Cellular sensitization was detected by the MEM-test using 3M KCl-extracts of fetuses from man and mouse and of different metamorphic stages of the water frog (tadpoles) and the carp (yolk sack-bearing stage). The sensitization demonstrated was independent on the type of tumor tested. The occurrence of active components in the extracts was correlated with certain stages of individual development of water frog and carp. The evidence of these components in species of several classes of vertebrates confirms the assumption of phylogenetically conserved structures.

[Demonstration and characterization of tumor-associated antigenic components from cell membranes of a UV-induced murine sarcoma using the MEM technic]. / Zum Nachweis und zur Charakterisierung tumorassoziierter antigener Komponenten aus Zellmembranen eines UV-Licht-induzierten Sarkoms der Maus mit Hilfe der MEM-Technik.

Purification of tumour-associated antigenic material from the ascites sarcoma cells was attempted by extraction with 3 M KCl and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The antigenic activity was assayed in the macrophage-electrophoretic-mobility (MEM) test. Extraction of whole tumour cells with 3 M KCl results in preparations with low antigenic activity which on SDS-PAGE show a very heterogenous composition of more than 20 protein bands. On comparison of the antigenic activity of different subcellular fractions obtained by differential centrifugation of the homogenate, the antigenic activity could be alloted to the cell membrane fraction. With this cell membrane fraction, three extraction media -- 3 M KCl, 2% Triton X-100, and 5% sodium cholate -- were tested for their ability to selectively extract the antigenic membrane proteins. The Triton X-100 treatment solubilized the greatest amount of membrane protein. Both Triton X-100 and sodium cholate, however, produced very heterogeneous protein extracts. In contrast, 3 M KCl selectively extracted three membrane components: a glycoprotein of high molecular weight and two low-molecular-weight carbohydrate-free proteins. Removal of the KCl in the presence of Triton X-100 precipitates the carbohydrate-free proteins, while the glycoprotein remains in solution. Testing the components of the KCl-extracts in the MEM-test after isolation by preparative SDS-PAGE revealed antigenic activity only with the glycoprotein component.

Differences of the in vitro reactivity of lymphocytes from normal and persistently lymphocytotic cows to enzootic bovine leukosis tumour membrane extracts as detected with the macrophage-electrophoretic mobility test.

The response of peripheral blood lymphocytes from normal and persistently lymphocytotic cows to cell membranes and sodium cholate-extracted membrane proteins from normal and tumourous lymph nodes was tested with the macrophage-electrophoretic-mobility test with guinea pig peritoneal macrophages as indicator cells. Lymphocytes from 13 cows with persistent lymphocytosis (PL) showed a positive reaction with cholate extracts from tumour cell membranes. On the other hand, there was a negative reaction with corresponding extracts from normal lymph nodes for 11 animals. 4 of 10 hematologically normal cows also showed a positive reaction against tumour cell membrane extracts. One of these, however, thereafter developed antibodies against BLV glycoprotein gp 51. Using tumour cell membrane preparations as antigen, the 4 cows with PL tested also exhibited a positive reaction, whereas none of 3 hematologically normal cows showed any reactivity. As efforts to detect the main antigenic proteins of BLV (gp 51, p 24) in detergent extracts of tumour cell membranes proved unsuccessful, these findings are thought to provide evidence for the existence of a common non-viral antigen on cell membranes of bovine leukosis tumour cells.